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Protein microarrays and biomarkers of infectious disease.

Natesan M, Ulrich RG - Int J Mol Sci (2010)

Bottom Line: Protein microarrays are powerful tools that are widely used in systems biology research.Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods.Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA; E-Mail: rulrich@bioanalysis.org.

ABSTRACT
Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.

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Related in: MedlinePlus

Measuring probe interactions with microarrayed proteins. Top: Detecting binding events using a fluorescently-labeled secondary probe, such as an antibody, and the resulting laser-scanned image. Bottom: Label-free interactions detected by surface plasmon resonance, resulting in a sensorgram of binding kinetics.
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f1-ijms-11-05165: Measuring probe interactions with microarrayed proteins. Top: Detecting binding events using a fluorescently-labeled secondary probe, such as an antibody, and the resulting laser-scanned image. Bottom: Label-free interactions detected by surface plasmon resonance, resulting in a sensorgram of binding kinetics.

Mentions: Large-scale genome sequencing projects first advanced knowledge of the theoretical composition of proteomes and led to the development of DNA microarrays for studying gene transcription at the organism-scale. Using genome sequence data to guide the direct examination of proteomes then enabled the study of host-pathogen interactions occurring beyond the level of gene transcription. It soon became evident that direct correlations between gene expression and protein abundance were rare [1,2], driving the development of new approaches to study complex proteomes. Protein microarrays are ideally suited to serve this purpose and have enormous potential applications in biomarker discovery, diagnosis, vaccine development, and drug discovery for infectious diseases. Fluorescence-based detection of protein interactions is similar to gene array methods, and data analysis often employs approaches previously developed for genome and transcription studies. The number of proteins that can be printed on a single microarray surface also approaches the same upper limits as nucleic-acid based systems. Multiplexing of fluorescent probes is limited by the ability to separate signals of overlapping emission spectra. Generally, pair-wise comparisons of 2–3 differentially labeled, experimental and control samples can be analyzed. For example, serum IgG and IgM binding to arrayed proteins can be independently probed by fluorescently labeled secondary antibodies, and detected by a confocal laser scanner (such as Genepix, Molecular Devices, Sunnyvale, CA, USA). A complete high-throughput screening of thousands of interactions can be performed accurately and rapidly (Figure 1). Direct labeling of probes may also be used, though structure and function may be adversely affected. Most label-free techniques [3] provide real-time measurements and in some cases yield kinetics (Figure 1), providing further insight into molecular interactions. Surface plasmon resonance (SPR) [4], nanowire surfaces [5], and mass spectrometry [6] are examples of label-free methods that are applied in the field of protein microarrays. Of these, only a limited number of SPR-based instruments are currently available for analysis of complex protein microarrays. SPR imaging (SPRi) is a more recent and promising development [3,7]. While planar arrays that use slides or chips for immobilizing capture molecules are most common, suspension arrays based on microbeads have many important applications. The xMAP technology developed by Luminex Corporation (Austin, TX, USA) uses 5.6 micrometer polymer beads infused with two fluorescent dyes at different ratios to yield up to 100 distinct bead sets. The binding measurements are performed by flow cytometry with two lasers, one for the identification of the bead and the other for the sample. Bead-based arrays are very useful for standardizing assays, with an upper limit of 80 independent immobilized probes.


Protein microarrays and biomarkers of infectious disease.

Natesan M, Ulrich RG - Int J Mol Sci (2010)

Measuring probe interactions with microarrayed proteins. Top: Detecting binding events using a fluorescently-labeled secondary probe, such as an antibody, and the resulting laser-scanned image. Bottom: Label-free interactions detected by surface plasmon resonance, resulting in a sensorgram of binding kinetics.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100839&req=5

f1-ijms-11-05165: Measuring probe interactions with microarrayed proteins. Top: Detecting binding events using a fluorescently-labeled secondary probe, such as an antibody, and the resulting laser-scanned image. Bottom: Label-free interactions detected by surface plasmon resonance, resulting in a sensorgram of binding kinetics.
Mentions: Large-scale genome sequencing projects first advanced knowledge of the theoretical composition of proteomes and led to the development of DNA microarrays for studying gene transcription at the organism-scale. Using genome sequence data to guide the direct examination of proteomes then enabled the study of host-pathogen interactions occurring beyond the level of gene transcription. It soon became evident that direct correlations between gene expression and protein abundance were rare [1,2], driving the development of new approaches to study complex proteomes. Protein microarrays are ideally suited to serve this purpose and have enormous potential applications in biomarker discovery, diagnosis, vaccine development, and drug discovery for infectious diseases. Fluorescence-based detection of protein interactions is similar to gene array methods, and data analysis often employs approaches previously developed for genome and transcription studies. The number of proteins that can be printed on a single microarray surface also approaches the same upper limits as nucleic-acid based systems. Multiplexing of fluorescent probes is limited by the ability to separate signals of overlapping emission spectra. Generally, pair-wise comparisons of 2–3 differentially labeled, experimental and control samples can be analyzed. For example, serum IgG and IgM binding to arrayed proteins can be independently probed by fluorescently labeled secondary antibodies, and detected by a confocal laser scanner (such as Genepix, Molecular Devices, Sunnyvale, CA, USA). A complete high-throughput screening of thousands of interactions can be performed accurately and rapidly (Figure 1). Direct labeling of probes may also be used, though structure and function may be adversely affected. Most label-free techniques [3] provide real-time measurements and in some cases yield kinetics (Figure 1), providing further insight into molecular interactions. Surface plasmon resonance (SPR) [4], nanowire surfaces [5], and mass spectrometry [6] are examples of label-free methods that are applied in the field of protein microarrays. Of these, only a limited number of SPR-based instruments are currently available for analysis of complex protein microarrays. SPR imaging (SPRi) is a more recent and promising development [3,7]. While planar arrays that use slides or chips for immobilizing capture molecules are most common, suspension arrays based on microbeads have many important applications. The xMAP technology developed by Luminex Corporation (Austin, TX, USA) uses 5.6 micrometer polymer beads infused with two fluorescent dyes at different ratios to yield up to 100 distinct bead sets. The binding measurements are performed by flow cytometry with two lasers, one for the identification of the bead and the other for the sample. Bead-based arrays are very useful for standardizing assays, with an upper limit of 80 independent immobilized probes.

Bottom Line: Protein microarrays are powerful tools that are widely used in systems biology research.Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods.Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USA; E-Mail: rulrich@bioanalysis.org.

ABSTRACT
Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.

Show MeSH
Related in: MedlinePlus